The need for new and effective antibiotics is well known, promoted as it is, not just in the annals of science, but in the popular press as well. Resistance mechanisms have rendered many antibiotics ineffective while drug discovery efforts of the industry have turned to more profitable targets. Recently, the CDC reported a death in Nevada caused by a Klebsiella pneumonia strain resistant to all 26 antibiotics in the US arsenal of potential treatments.1 These 'superbugs' remain unusual, however, and the post-antibiotic era is not a foregone conclusion. Properly managed treatment strategies using new antibiotics with novel mechanisms of action hold the promise of antibiotic efficacy well into the future. We propose here a discovery program to identify new antibiotics with the novel mechanisms of action. Our program seeks to discover new antibiotics from natural products, the major source of existing antimicrobial therapeutics. Natural products are an outstanding source of new chemotypes bearing antibiotic properties. Consider that in the 25 years between 1981-2006, 74 new antibiotics were registered as either native natural products, or natural product derivatives, while only 23 totally synthetic compounds were discovered. Indeed, 2 of the 3 new classes of antibiotics discovered since 1970 are natural products (daptomycin and mupirocin). The exquisite selectivity and unimaginable diversity of natural products is no accident; it is designed by nature for survival purposes that are largely driven by microbial warfare. Our discovery pipeline includes a new class of biofilm-selective antibiotics derived from one of our natural product sources, a cold-water sponge. Mechanism-of-action (MOA) studies of these spongian diterpenes in Aim 3 may provide critically needed insights into biofilm disruption. The pipeline also includes high priority antibiotic extracts which we aim to characterize chemically (Aim 1) and biologically (Aim 2) to identify new natural product hits for progression to MOA and related comprehensive advanced biological screening (Aim 3). Further, we aim to keep the pipeline full through primary screening (Aim 2) of new antibiotic screening samples (Aim 1) derived from largely unstudied biological sources, including cold-water marine invertebrates, marine fungi and actinomycetes, and the comprehensive NCI pre-fractionated library of plant and marine natural product extracts. Our project brings innovation in culture miniaturization and elicitation of silent biosynthetic pathways to maximize screening throughput, and a chromatographic technique to reduce effort lost in chemotype re- discovery. All chemodiversity will be evaluated in a high-throughput assay using the clinically relevant ESKAPE pathogens. Hits will be evaluated for cytotoxicity, with those displaying favorable characteristics advancing to comprehensive secondary screening.